Method of producing macrolide compound

ABSTRACT

The present invention provides a novel method of producing the 12-membered ring macrolide compound 11107D having an antitumor activity by biological transformation. Starting material which is the 12-membered ring macrolide compound 11107B represented by the formula (I) is incubated in the presence of a strain belonging to the genus  Mortierella , the genus  Streptomyces  or the family Micromonosporaceae (for example,  Streptomyces  sp. AB-1704 strain (FERM BP-8551)), each of which has the ability of transforming the 12-membered ring macrolide compound 11107B into a 11107D substance represented by the formula (II), or a preparation of its cultured mycelia and oxygen, and then 11107D substance which is a target material is collected from the treating solution.

TECHNICAL FIELD

The present invention relates to a method of producing the 12-memberedring macrolide compound 11107D having an antitumor activity bybiological transformation and to a novel strain used for the production.

PRIOR ART

The 12-membered ring macrolide compound 11107D is a 12-membered ringmacrolide compound having an excellent antitumor activity and wasdiscovered together with a 11107B substance from a culture product of aStreptomyces sp. Mer-11107 strain (see WO-A02/060890) . The 11107Dsubstance corresponds to a 16-position hydroxide body of the 11107B. Theproductivity of the 11107D substance is inferior to that of the 11107Bsubstance and it has been therefore desired to establish an efficientproduction method.

DISCLOSURE OF THE INVENTION

The purpose of the present invention is to provide a novel method ofproducing the macrolide compound 11107D by using the macrolide compound11107B as starting material by a biological transformation method.

The inventors of the present invention have made a trial to selectmicroorganisms capable of transforming the 16-position hydrogen atom tohydroxyl group of the macrolide compound 11107B by screening from a widerange of microorganism groups to solve the above problem, and as aresult, found that a strain belonging to the genus Mortierellaclassified into the filamentous fungi, a strain belonging to the genusStreptomyces classified into actinomycetes and a strain belonging to thefamily Micromonosporaceae likewise classified into actinomycetes havethe above-mentioned transforming function, to complete the presentinvention.

Accordingly, the present invention relates to the following (1) to (3).

-   (1) A method of producing the macrolide compound 11107D represented    by the formula (II):    wherein the macrolide compound 11107D is produced from the macrolide    compound 11107B represented by the formula (I):    by a biological transformation method, which comprises the following    processes (A) and (B):

(A) a process of incubating the macrolide compound 11107B represented bythe formula (I) in the presence of a strain having an ability ofconducting the above-mentioned biological transformation method andbelonging to the genus Mortierella, the genus Streptomyces or the familyMicromonosporaceae or a preparation of its cultured mycelia; and

(B) a process of collecting the macrolide compound 11107D represented bythe formula (II) from the incubated solution obtained in the step (A).

-   (2) The production method according to the above (1), wherein the    strain belonging to the genus Mortierella is Mortierella sp. F-1529    strain (FERMBP-8547) or F-1530 strain (FERMBP-8548)-   (3) The production method according to the above (1), wherein the    strain belonging to the genus Streptomyces is Streptomyces sp.    AB-1704 strain (FERMBP-8551), A-1544 strain (FERMBP-8446) or A-1545    strain (FERM BP-8447).-   (4) The production method according to the above (1), wherein the    strain belonging to the family Micromonosporaceae is AB-1896 strain    (FERM BP-8550).-   (5) Streptomyces sp. AB-1704 strain (FERM BP-8551) having the    ability of transforming the macrolide compound 11107B represented by    the formula (I) into the macrolide compound 11107D represented by    the formula (II).-   (6) Mortierella sp. F-1529strain (FERMBP-8547) or F-1530 strain    (FERM BP-8548) having the ability of transforming the macrolide    compound 11107B represented by the formula (I) into the macrolide    compound 11107D represented by the formula (II).-   (7) AB-1896 strain (FERM BP-8550) having the ability of transforming    the macrolide compound 11107B represented by the above formula (I)    into the macrolide compound 11107D represented by the above formula    (II).

DETAILED DESCRIPTION OF THE INVENTION

In the biological transformation method of the present invention, anymicroorganisms belonging to the genus Mortierella, the genusStreptomyces or the family Micromonosporaceae may be used regardless ofthe type of species and strain insofar as it has the ability totransform the macrolide compound 11107B represented by the above formula(I) into the macrolide compound 11107D represented by the above formula(II). However, preferable examples of the microorganisms may include theMortierella sp. F-1529 strain and F-1530 strain belonging to the genusMortierella, the Streptomyces sp. AB-1704 strain, A-1544 strain andA-1545 strain belonging to the genus Streptomyces and the AB-1896 strainbelonging to the family Micromonosporaceae, each has isolated from thesoil.

The Mer-11107 strain was deposited as FERM P-18144 at the NationalInstitute of Bioscience and Human-Technology Agency of IndustrialScience and Technology (1-3, Higashi 1-chome Tsukuba-shi, Ibaraki-ken305-8566 Japan) as of Dec. 19, 2000, and then transferred toInternational Deposit FERMBP-7812 at International Patent OrganismDepositary (IPOD) National Institute of Advanced Industrial Science andTechnology (Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi,Ibaraki-ken 305-8566 Japan) as of November 27, 2001.

The Mortierella sp. F-1529 strain was internationally deposited atInternational Patent Organism Depositary National Institute of AdvancedIndustrial Science and Technology (Tsukuba Central 6, 1-1, Higashi1-Chome, Tsukuba-shi, Ibaraki-ken 305-8566 Japan) as of Nov. 12, 2003 asFERM BP-8547. Also, the Mortierella sp. F-1530 strain wasinternationally deposited at International Patent Organism DepositaryNational Institute of Advanced Industrial Science and Technology(Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken305-8566 Japan) as of Nov. 12, 2003 as FERM BP-8548.

The Streptomyces sp. AB-1704 strain was deposited at InternationalPatent Organism Depositary National Institute of Advanced IndustrialScience and Technology (Tsukuba Central 6, 1-1, Higashi 1-Chome,Tsukuba-shi, Ibaraki-ken 305-8566 Japan) as FERM P-18999 as of September5, 2002, and then transferred to International Deposit FERM BP-8551 asof Nov. 12, 2003, at International Patent Organism Depositary (IPOD)National Institute of Advanced Industrial Science and Technology(Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken305-8566 Japan). Also, the A-1544 strain and A-1545 strain weredeposited at International Patent Organism Depositary National Instituteof Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1,Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken 305-8566 Japan) as FERMP-18943 and FERM P-18944 as of Jul. 23, 2002, and then transferred toInternational Deposit FERM BP-8446 and FERM BP-8447 as of July 30, 2003respectively, at International Patent Organism Depositary (IPOD)National Institute of Advanced Industrial Science and Technology(Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken305-8566 Japan).

The AB-1896 strain belonging to the family Micromonosporaceae wasinternationally deposited at International Patent Organism DepositaryNational Institute of Advanced Industrial Science and Technology(Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken305-8566 Japan) as FERM-BP 8550 as of Nov. 12, 2003.

The taxonomical properties of the above strains are as follows.

(The Taxonomical Properties of the F-1529 Strain)

(1) Morphological Characteristics

On each plate of oatmeal agar (hereinafter abbreviated as OA as the casemay be), malt agar (2% malt extract+1.5% agar: hereinafter abbreviatedas MEA as the case may be) and potato dextrose agar (hereinafterabbreviated as PDA as the case may be), the colony had a floccose formand the color of the hyphae was white, exhibiting white (1A-1) tone. Asto the growth when the strain was cultured at 25° C. for one week, thecolonies reached 75 mm in diameter on the OA plate, 75 to 80 mm indiameter on the MEA plate and 75 to 80 mm in diameter on the PDA plate.The colony had a zonate shape. Neither backside coloring nor theproduction of soluble pigment was not observed. The the color tone wasdescribed according to “Methuen Handbook of Colour (Kornerup & Wanscher,1978)”.

As a result of the observation using an optical microscope, thevegetative hyphae were colorless, had a smooth surface, were providedwith no septum and had a width of 4 to 5 μm. A swelled structure likethat of a thick wall spore having a spherical form and a size of about26.5 to 33 μm was observed in a part of the hypahe. In the culture mediato be subjected to the test, any structure that seems to be a genitalorgan was not formed even by culturing for a term not exceeding 3 weeks.

(2) 18S rRNA Gene Analysis

A mycelia of the F-1529 strain cultured on an agar plate was subjectedto DNA extraction using Fast Prep FP120 (manufactured by Q-BIO gene) andFast DNA Kit (manufactured by Q-BIO gene). PCR was practiced usingpuRetaq Ready-To-Go PCR beads (manufactured by Amersham Biosciences) andPCR primers NS1 and NS8 shown in Tables 1 and 2. The PCR products wererefined using QIAquick PCR Purification Kit (manufactured by QIAGEN) andthen treated with ABI Prism BigDye Terminator Kit (manufactured byApplied Biosystems). As the sequencing primers NS1, NS2, NS3, NS4, NS5,NS6, NS7 and NS8 shown in Tables 1 and 2 were used. The reactionproducts were refined using a Dye EX2.0 Spin Kit (manufactured byQIAGEN) and subjected to sequence analysis using an ABI PRISM 3100Genetic Analyzer (manufactured by Applied Biosystems). Then, thesequenced fragments were combined together by using an Auto Assembler(manufactured by Applied Biosystems) to obtain the full lengthnucleotide sequence. TABLE 1 Primers used to determine nucleotidesequence of 18s rRNA gene (forward direction) Sequence No. Name Sequence1 NS1 5′-gtagtcatatgcttgtct-3′ 2 NS3 5′-gcaagtctggtgccagcagcc-3′ 3 NS55′-aacttaaaggaattgacggaag-3′ 4 NS7 5′-gaggcaataacaggtctgtgatg-3′

TABLE 2 Primers used to determine nucleotide sequence of 18s rRNA gene(reverse direction) Sequence No. Name Sequence 5 NS25′-cgttcagaccacggtcgtcgg-3′ 6 NS4 5′-ttgaatttccttaactgccttc-3′ 7 NS65′-ctccgttattgtccagacactac-3′ 8 NS8 5′-aggcatccacttggacgcct-3′Thus obtained 18S rRNA gene of the strain has a nucleotide sequencedescribed in Sequence No. 9.

The DNA sequence of a known strain was obtained from Japan DNA Data Bank(http://www.ddbj.nig.ac.jp/) to examine the homology of the 18S rRNAgenes. As a result, this 18S rRNA gene had 99% (upstream 803 bases)homology with the 18S rRNA gene of Mortierella hyalina (GenBank,accession no. AY157493), 98% (full length) homology with the 18S rRNAgene of Mortierella chlamydospora (GenBank accession No. AF157143) and98% (full length) homology with the 18S rRNA gene of Mortierellamultidivaricata (GenBank accession No. AF157144).

From the above mycological properties, the inventors of the presentinvention determined that this strain belongs to the genus Mortierella.

Taxonomical Properties of the F-1530 Strain

Morphological Characteristics

On each plate of oatmeal agar, malt agar and potato dextrose agar, thecolony had a cottony form and the color of the hyphae was white,exhibiting white (1A-1) tone. As to the growth when the strain wascultured at 25° C. for one week, the strain reached 80 to 85 mm indiameter on the OA plate, 85 mm in diameter on the MEA plate and 85 mmin diameter on the PDA plate. The colony had a zonate shape. Neitherbackside coloring nor the production of soluble pigment was notobserved. The color tone was described according to “Methuen Handbook ofColour (Kornerup & Wanscher, 1978)).

As a result of observation using an optical microscope, vegetativehyphae were colorless, had a smooth surface, were provided with noseptum and had a width of 2.5 to 5 μm. As welled structure like that ofa thick wall spore, having a spherical form and a size of about 10 μm isobserved in a part of the hyphae. In the culture media to be subjectedto the test, any structure that seems to be a genital organ was notformed even by culturing for a term not exceeding 3 weeks.

(2) 18S rRNA Gene Analysis

The 18S rRNA gene of the F-1530 strain was analyzed in the same manneras in the case of the F-1529 strain. Thus obtained 18S rRNA gene of theF-1530 strain had a nucleotide sequence described in Sequence No. 10.

The DNA sequence of a known strain was obtained from Japan DNA Data Bank(http://www.ddbj.nig.ac.jp/) to examine the homology of the 18S rRNAgenes. As a result, this 18S rRNA gene had 100% (upstream 803 bases)homology with the 18S rRNA gene of Mortierella hyalina (GenBank,accession no. AY157493), 98% (full length) homology with the 18S rRNAgene of Mortierella chlamydospora (GenBank accession No. AF157143) and98% (full length) homology with the 18S rRNA gene of Mortierellamultidivaricata (GenBank accession No. AF157144).

From the above-mentioned microbial characteristics, the presentinventors determined that the present strain belongs to the genusMortierella.

(The Taxonomical Properties of the AB-1704 Strain)

(1) Morphological Characteristics

Rectiflexibiles type aerial hyphae were extended from vegetative hyphaein this strain. Spore chains consisting of about 20 to 50 of cylindricalspores were formed at the end of the matured aerial hyphae. The size ofthe spores was about 0.6 to 0.8×1.0 to 1.1 μm, the surface of the sporeswas smooth, and specific organs such as sporangium, sclerotium andflagellum were not observed.

(2) Cultural Characteristics on Various Media

Cultural characteristics of the strain after incubation at 28° C. fortwo weeks on various media are shown in Table 3. The color tone isdescribed by the color names and codes which are shown in theparentheses of Tresner's Color wheels. TABLE 3 Color of vegetativeSoluble Medium Growth Aerial hyphae hyphae pigment Yeast extract - GoodThick Nude tan None malt extract agar Ivory (2db) (4gc) (ISP-2) Oatmealagar Good Abundant Light melon None (ISP-3) Ivory (2db) yellow (3ea)Inorganic salts - Good Thick Cork tan None starch agar Putty-Ivory (4ie)(ISP-4) (1½ec-2db) Glycerol - Good Thick Nude tan None asparagine agarParchment (4gc) (ISP-5) (1½db) Peptone-yeast Good Abundant Light melonNone extract - iron agar White (a) yellow (ISP-6) (3ea) Tyrosine agarGood Thick Nude tan None (ISP-7) Ivory (2db) (4gc)(3) Utilization of Various Carbon Sources

Various carbon sources were added to Pridham-Gottlieb agar and culturedat 28° C. for 2 weeks. The growth of the strain is shown in Table 4.TABLE 4 D-glucose + inositol − L-arabinose ± L-rhamnose + D-xylose +D-mannitol + D-fructose + raffinose − sucrose ±(+: positive, ±: slightly positive, −: negative)(4) Various Physiological Properties

Various physiological properties of the present strain are as follows.

-   (a) Range of growth temperature (yeast extract-malt extract agar,    incubation for 2 weeks) 5° C. to 33° C.-   (b) Range of optimum growth temperature (yeast extract-malt extract    agar, incubation for 2 weeks) 15° C. to 33° C.-   (c) Liquefaction of gelatin (glucose-peptone-gelatin medium)    positive-   (d) Coagulation of milk (skim milk medium) positive-   (e) Peptonization of milk (skim milk medium) positive-   (f) Hydrolysis of starch (inorganic salts-starch agar) positive-   (g) Formation of melanoid pigment (peptone-yeast extract-iron agar)    negative, (tyrosine agar) negative-   (h) Production of hydrogen sulfide (peptone-yeast extract-iron agar)    negative-   (i) Reduction of nitrate (broth containing 0.1% potassium nitrate)    positive-   (j) Sodium chloride tolerance (yeast extract-malt extract agar,    incubation for 2 weeks) grown at a salt content of 7% or less    (5) Chemotaxonomy

LL-diaminopimelic acid was detected from the cell wall of the presentstrain.

From the above-mentioned microbial characteristics, the presentinventors determined that the present strain belongs to the genusStreptomyces.

(The Taxonomical Properties of A-1544 Strain)

(1) Morphological Characteristics

Spira type aerial hyphae were extended from vegetative hyphae in thisstrain. Spore chains consisting of about 10 to 20 of cylindrical sporeswere formed at the end of the matured aerial hyphae. The size of thespores was about 1.0×1.2 to 1.94 μm, the surface of the spores wasspiny, and specific organs such as sporangium, sclerotium and flagellumwere not observed.

(2) Cultural Characteristics on Various Media

Cultural characteristics of the strain after incubation at 28° C. fortwo weeks on various media are shown in Table 5. The color tone isdescribed by the color names and codes which are shown in theparentheses of Tresner's Color wheels. TABLE 5 Color of vegetativeSoluble Medium Growth Aerial hyphae hyphae pigment Yeast extract - GoodThick Light melon None malt extract Silver gray yellow agar (ISP-2)(3fe) (3ea) Oatmeal agar Good Abundant Light melon None (ISP-3) Lightgray-Silver yellow gray (d-3fe) (3ea) Inorganic salts - Good AbundantLight melon None starch agar Silver gray (3fe) yellow (ISP-4) (3ea)Glycerol - Good Abundant Light melon None asparagine agar Ashes (5fe)yellow (ISP-5) (3ea) Peptone-yeast Good None Light melon Pale extract -iron yellow blackish agar (ISP-6) (3ea) brown Tyrosine agar GoodAbundant Light melon None (ISP-7) Covert gray (2fe) yellow (3ea)(3) Utilization of Various Carbon Sources

Various carbon sources were added to Pridham-Gottlieb agar and culturedat 28° C. for 2 weeks. The growth of the strain is shown in Table 6.TABLE 6 D-glucose + inositol − L-arabinose + L-rhamnose + D-xylose +D-mannitol + D-fructose + raffinose − sucrose −(+: positive, −: negative)(4) Various Physiological Properties

Various physiological properties of the present strain are as follows.

-   (a) Range of growth temperature (yeast extract-malt extract agar,    incubation for 2 weeks) 15° C. to 41° C.-   (b) Range of optimum growth temperature (yeast extract-malt extract    agar, incubation for 2 weeks) 20° C. to 37° C.-   (c) Liquefaction of gelatin (glucose-peptone-gelatin medium)    positive-   (d) Coagulation of milk (skim milk medium) positive-   (e) Peptonization of milk (skim milk medium) positive-   (f) Hydrolysis of starch (inorganic salts-starch agar) positive-   (g) Formation of melanoid pigment (peptone-yeast extract-iron agar)    positive, (tyrosine agar) negative-   (h) Production of hydrogen sulfide (peptone-yeast extract-iron agar)    positive-   (i) Reduction of nitrate (broth containing 0.1% potassium nitrate)    negative-   (j) Sodium chloride tolerance (yeast extract-malt extract agar,    incubation for 2 weeks) grown at a salt content of 7% or less    (5) Chemotaxonomy

LL-diaminopimelic acid was detected from the cell wall of the presentstrain.

From the above-mentioned microbial characteristics, the presentinventors determined that the present strain belongs to the genusStreptomyces.

(The Taxonomical Properties of A-1545 Strain)

(1) Morphological Characteristics

Rectiflexibiles type aerial hyphae were extended from vegetative hyphaein this strain. Spore chains consisting of about 50 of spores wereformed at the end of the matured aerial hyphae. The size of the sporeswas about 0.8×1.0 μm, the surface of the spores was smooth, and specificorgans such as sporangium, sclerotium and flagellum were not observed.

(2) Cultural Characteristics on Various Media

Cultural characteristics of the strain after incubation at 28° C. fortwo weeks on various media are shown in Table 7. The color tone isdescribed by the color names and codes which are shown in theparentheses of Tresner's Color wheels. TABLE 7 Color of vegetativeSoluble Medium Growth Aerial hyphae hyphae pigment Yeast extract - GoodAbundant Grayish Light melon None malt extract yellowish pinkyellow-Nude agar (ISP-2) (5cb) tan (3ea-4gc) Oatmeal agar Moderate ThinGrayish Pearl pink None (ISP-3) yellowish pink (3ca) (5cb) Inorganicsalts - Good Thin Grayish Light Ivory None starch agar yellowish pink(2ca) (ISP-4) (5cb) Glycerol - Good Abundant Grayish Pearl pink Noneasparagine agar yellowish pink (3ca) (ISP-5) (5cb) Peptone-yeastModerate None Light melon None extract - iron yellow agar (ISP-6) (3ea)Tyrosine agar Good Abundant Grayish Light melon None (ISP-7) yellowishpink yellow (5cb) (3ea)(3) Utilization of Various Carbon Sources

Various carbon sources were added to Pridham-Gottlieb agar and culturedat 28° C. for 2 weeks. The growth of the strain is shown in Table 8.TABLE 8 D-glucose + inositol ± L-arabinose + L-rhamnose + D-xylose +D-mannitol + D-fructose + raffinose + sucrose −(+: positive, ±: slightly positive, −: negative)(4) Various Physiological Properties

Various physiological properties of the present strain are as follows.

-   (a) Range of growth temperature (yeast extract-malt extract agar,    incubation for 2 weeks) 10° C. to 37° C.-   (b) Range of optimum growth temperature (yeast extract-malt extract    agar, incubation for 2 weeks) 20° C. to 33° C.-   (c) Liquefaction of gelatin (glucose-peptone-gelatin medium)    negative-   (d) Coagulation of milk (skim milk medium) positive-   (e) Peptonization of milk (skim milk medium) positive-   (f) Hydrolysis of starch (inorganic salts-starch agar) positive-   (g) Formation of melanoid pigment (peptone-yeast extract-iron agar)    negative, (tyrosine agar) negative-   (h) Production of hydrogen sulfide (peptone-yeast extract-iron agar)    positive-   (i) Reduction of nitrate (broth containing 0.1% potassium nitrate)    negative-   (j) Sodium chloride tolerance (yeast extract-malt extract agar,    incubation for 2 weeks) grown at a salt content of 7% or less    (5) Chemotaxonomy

LL-diaminopimelic acid was detected from the cell wall of the presentstrain.

From the above-mentioned microbial characteristics, the presentinventors determined that the present strain belongs to the genusStreptomyces.

(The Taxonomical Properties of AB-1896 Strain)

(1) Morphological Characteristics

The AB-1896 strain showed good or moderate growth on the culture mediaused to identify the strain at 28° C. for 7 to 14 days. No hypha wasobserved during the cultures and one spore was observed on eachvegetative hyphae. The spore had a sphere form and the size thereof wasabout 0.8 to 0.9 μm, and the surface of the spores was warty. Specificorgans such as sporangium, sclerotium and flagellum were not observed.

(2) Cultural Characteristics on Various Media

Cultural characteristics of the strain after incubation at 28° C. fortwo weeks on various media are shown in Table 9. The color tone isdescribed by the color names and codes which are shown in theparentheses of Tresner's Color wheels. TABLE 9 Color of vegetativeSoluble Medium Growth hyphae pigment Yeast extract - Good Bisque Nonemalt extract agar Berver (3li) (3ec) (ISP-2) Oatmeal agar Moderate Lightmelon None (ISP-3) Cork tan (4ie) yellow (3ea) Inorganic salts - GoodLight melon None starch agar Berver (3li) yellow (ISP-4) (3ea)Glycerol - Moderate Pearl pink None asparagine agar Light olive (3ca)(ISP-5) drab (1li) Peptone-yeast Good Light melon None extract - ironBerver (3li) yellow agar (ISP-6) (3ea) Tyrosine agar Moderate Pearl pinkNone (ISP-7) Light olive (3ca) gray (1½ge)(3) Utilization of Various Carbon Sources

Various carbon sources were added to Pridham-Gottlieb agar and culturedat 28° C. for 2 weeks. The growth of the strain is shown in Table 10.TABLE 10 D-glucose + inositol − L-arabinose + L-rhamnose − D-xylose +D-mannitol − D-fructose + raffinose + sucrose +(+: positive, −: negative)(4) Various Physiological Properties

Various physiological properties of the present strain are as follows.

(a) Range of growth temperature (yeast extract-malt extract agar,incubation for 2 weeks) 20° C. to 41° C.

(b) Range of optimum growth temperature (yeast extract-malt extractagar, incubation for 2 weeks) 25° C. to 37° C.

(c) Liquefaction of gelatin (glucose-peptone-gelatin medium) negative

(d) Coagulation of milk (skim milk medium) positive

(e) Peptonization of milk (skim milk medium) positive

(f) Hydrolysis of starch (inorganic salts-starch agar) positive

(g) Formation of melanoid pigment (peptone-yeast extract-iron agar)negative, (tyrosine agar) negative

(h) Production of hydrogen sulfide (peptone-yeast extract-iron agar)negative

(i) Reduction of nitrate (broth containing 0.1% potassium nitrate)positive

(j) Sodium chloride tolerance (yeast extract-malt extract agar,incubation for 2 weeks) not grown at a salt content of 4%

(5) Chemotaxonomy

Meso-type diaminopimelic acid was detected from the cell wall of theAB-1896 strain. As major structural sugars of whole mycelia, xylose andmannose were detected. The type of acyl inpeptide glycan of the cellwall was a glycolyl type. Mycolic acid was not detected. As majormenaquinone components, MK-9(H₄), MK-9(H₆), MK-10(H₄) and MK-10(H₆) weredetected.

(6) Analysis of the 16S rRNA Gene

A culture broth of the AB-1896 strain was collected and then subjectedto DNA extraction using Fast DNA kit (manufactured by Q-BIO gene). PCRwas carried out in a reaction condition of 96° C./20 seconds, 50° C./20seconds and 72° C./one minute in 30 cycles in total. As the primers,9F(5′-GTGTTTGATCCTGGCTCAG-3′) (sequence No. 11) and 536R(5′-GTATTACCGCGGCTGCTG-3′) (sequence No. 12) were used. The PCR productwas refined using a MinElute PCR Purification Kit (manufactured byQIAGEN) to provide a sequencing sample.

The sequencing was carried out using an ABI PRISM 310 Genetic Analyzer(manufactured by Applied Biosystems) and a BigDye Terminator kitaccording to their standard protocols. As the primers, 9F and 536R wereused.

Thus obtained about 500 nucleotide sequenece on the 5′ terminal side ofthe 16s rRNA gene of the present strain is described in the sequence no.13.

The DNA sequences of known strains were obtained from Japan DNA DataBank (http://www.ddbj.nig.ac.jp/) to examine the homology of 400 to 500bases on the 5′ terminal side of the 16s rRNA genes. As a result, this16s rRNA gene had 98% homology with the 16s rRNA gene of Micromonosporasp. DSM44396 (GenBank, accession no. AJ560637), 98% homology with a geneof Micromonospora purpureochromogenes (GenBank accession No. X92611),95% homologywith a gene of M. Chalcea IFO12135 (GenBank, accession no.D85489) which is a type strain of genus Micromonospora and 95% homologywith a gene of Verrucosispora gifhornensis (GenBank, accession no.Y15523) which is a type strain of genus Verrucosispora.

Although the AB-1896 strain had almost the same characteristics as thegenus Micromonospora, it corresponded imperfectly to the genusMicromonospora in the point that arabinose was not detected but mannosewas detected as the major structural sugar of the whole mycelia. Also,the AB-1896 strain did not correspond perfectly to the genusVerrucosispora in the point that the spores peculiar in Verrucosisporagifhornensis were not observed on the medium used to identify thestrain. The present inventors of the present invention decided that theAB-1896 strain is actinomycetes belonging to the familyMicromonosporaceae in consideration of the above taxonomical properties.

According to the present invention, first in the process (A), themacrolide compound 11107B which is starting material (substrate) isincubated in the presence of the above strains or products prepared byits cultured mycelia and further in the presence of oxygen. Thistreatment may be carried out by adding the substrate in the culturebroth when culturing the above strains in an aerobic condition or, asthe case may be, by adding the substrate in a suspension solution of thecultured mycelia of the above strains as it is or of a product preparedby homogenizing these cells with flowing gas containing oxygen, forexample, air.

The substrate may be added to the culture broth before culturing or whena fixed time passes after the start of culturing. The above mycelia maybe produced by inoculating any one of the above strains into a mediumcontaining a nutrient and culturing aerobically. The culturing of thestrain for producing mycelia preparations or the culturing of the strainwhich is carried out in the situation where the substrate is added maybe performed according to a method of culturing general microorganismsfundamentally. However, in general, the culturing is preferably carriedout in an aerobic condition by, for example, flask shaking culture andtank culture according to liquid culture.

As the medium used for culturing, any medium may be used insofar as itcontains a nutrient which microorganisms belonging to the genusMortierella, the genus Streptomyces or the family Micromonosporaceae canutilize, and various synthetic media, semi-synthetic media and naturalmedia may be all utilized. As the medium composition, various carbonsources such as glucose, galactose, sucrose, maltose, fructose,glycerin, dextrin, starch, molasses and soybean oil may be used eitherindependently or in combinations.

As the nitrogen source, there can be used a single or a combination oforganic nitrogen sources such as pharma media, peptone, meat extract,soybean meal, fish meal, gluten meal, casein, dry yeast, aminoacid,yeast extract, NZ-caseandurea, and inorganic nitrogen sources such assodium nitrate and ammonium sulfate. Additionally, for example, therecan be added and used salts such as sodium chloride, potassium chloride,calcium carbonate, magnesium sulfate, sodium phosphate, potassiumphosphate copper sulfate, iron sulfate, manganese chloride or cobaltchloride; heavy metal salts; vitamins such as vitamin B or biotin; andinclusion agents such as cyclodextrins, if necessary. Further, whenfoaming is remarkable during culture, various defoaming agents can beappropriately added in the medium as necessary. When the defoaming agentis added, it is required to set at a concentration for not adverselyaffecting the production of an objective substance.

The culture condition can be appropriately selected within the range atwhich the microbial strain grows well and can produce theabove-mentioned substance. For example, the pH of a medium is about 5 to9, and preferably nearby neutral in general. The temperature offermentation is usually kept at 20° C. to 40° C. and preferably 24° C.to 30° C. The fermentation period is about 1 to 8 days, and usuallyabout 2 to 5 days. The above-mentioned fermentation conditions can besuitably changed in accordance with the kind and property ofmicroorganism used, external conditions and the like, and it is needlessto say that an optimum condition can be selected.

Also, the culture mycelia preparation is prepared by suspending myceliaseparated by centrifugation or filtration or homogenized in a propersolution after the culturing is finished. Examples of the solution usedfor the suspension of the mycelia include the above-mentioned medium orbuffer solutions such as tris-acetic acid, tris-hydrochloric acid,sodium succinate, sodium citrate, sodium phosphate and potassiumphosphate either singly or in combinations. The pH of the buffersolution is 5.0 to 9.0 and preferably 6.0 to 7.5.

The 11107B substance as the substrate may be added in a culture broth ora suspension solution of mycelia either as a powder as it is or as asolution dissolved in a water-soluble solvent, for example, ethanol,methanol, acetone or dimethylsulfoxide. The amount of the 11107Bsubstance added is preferably 50 to 5000 mg per 1 L of the culture brothin the case of the culture broth. After the addition of the substrate,procedures such as flask shaking or tank culture are conducted at 20 to31° C. for about 1 to 5 days to run a reaction in an aerobic condition,whereby the 11107B substance as the substrate can be converted into the11107D substance.

Next, in the process (B), the target 11107D substance is recovered fromthe incubated solution obtained in the above process (A). Suitablemethods are selected from various known purification methods which areusually used to isolate microorganism metabolites and used incombination to isolate the 11107D substance from the reaction mixture inthe process (A). For example, extraction by an organic solvent such asmethanol, ethanol, butanol, acetone, ethyl acetate, butyl acetate,chloroform or toluene; various kinds of ion-exchange chromatography; gelfiltration chromatography using Sephadex LH-20; the treatment ofadsorption and desorption by absorption chromatography using ahydrophobic adsorbing resin such as Diaion HP-20, active carbon orsilica gel, or thin layer chromatography; or high-performance liquidchromatography using a reverse phase column and so on may be usedindependently or in combinations or used repeatedly whereby the 11107Dsubstance can be separated and purified.

EXAMPLES

The present invention will be explained in more detail by way ofExamples, which are not intended to limit the scope of the presentinvention. In the following Examples, all designations of % indicateweight percentage (wt.%), unless otherwise noted.

Referential Example 1 Production of 11107B Substance as StartingMaterial

One loopful of the slant culture (ISP-2 medium) of Streptomyces sp.Mer-11107 strain (FERMBP-7812) was inoculated into a 500 mL Erlenmeyerflask containing 50 mL of seed medium (2.0% of glucose, 1.0% of soybeanmeal (ESUSAN-MEAT manufactured by Ajinomoto Co. Ltd.), 0.5% of yeastextract (manufactured by Oriental Yeast Co., Ltd.), 0.25% of sodiumchloride, 0.32% of calcium carbonate, pH 6.8 before sterilized), and itwas cultured at 28° C. for two days to give the first seed culturebroth. 0.1 mL of the culture broth was inoculated into a 500 mLErlenmeyer flask containing 100 mL of the same seed medium and it wascultured at 28° C. for one day to give the second seed culture broth.The second seed culture broth (800 mL) thus obtained was inoculated intoa 200 L tank containing 100 L of a production medium (5.0% of solublestarch, 0.8% of Pharmamedia, 0.8% of gluten meal, 0.5% of yeast extractand 0.1% of calcium carbonate, pH 6.8 before sterilized) and it wascultured for five days with flowing air and stirring in the followingconditions, to give a culture broth.

-   Culture temperature: 28° C.-   Agitation: 90 rpm-   Aeration: 1.0 vvm-   Internal pressure: 20 kPa

A part of the culture broth (10 L) thus obtained was extracted with 10 Lof 1-butanol, and then the resulting butanol layer was evaporated todryness, to give 100 g of crude active fraction. The crude activefraction was applied on Sephadex LH-20 (1500 mL; manufactured byPharmacia Co. Ltd. ), and eluted with tetrahydrofuran-methanol (1:1) asa solvent. An eluted fraction from 540 mL to 660 mL was concentrated todryness, to give a residue (660mg). The resulting residue was dissolvedin a mixture of ethyl acetate and methanol (9:1; v/v) and subjected tosilica gel column chromatography (WAKO GEL C-200, 50 g). The column waseluted with a mixture (2 L) consisting of n-hexane and ethyl acetate(1:9, v/v), the fractions eluted from 468 mL to 1260 mL were collected,evaporated to give 25 mg of a crude active fraction.

The obtained crude active fraction was subjected to preparative highperformance liquid chromatography (HPLC) under the following preparativeHPLC condition (A), and the fractions eluted at the retention time of 34minutes were collected. After removing acetonitrile, the respectivefractions were desalted by HPLC under the following preparative HPLCcondition (B) to give 11107B (Retentiontime: 37minutes, 6 mg).

Preparative HPLC Conditions A:

Column: YMC-PACK ODS-AM SH-343-5AM, φ20 mm×250 mm (manufactured by YMCCo.)

Temperature: room temperature

Flow rate: 10 mL/min.

Detection: 240 nm

Eluent: acetonitrile/0.15% aqueous potassium dihydrogenphosphate (pH3.5) (2:8 to 8:2, v/v, 0 to 50 min., linear gradient)

Preparative HPLC Conditions B:

Column: YMC-PACK ODS-AM SH-343-5AM, φ20 mm×250 mm (manufactured by YMCCo.)

Temperature: room temperature

Flow rate: 10 mL/min.

Detection: 240 nm

Eluent:-methanol/water (2:8 to 10:0, v/v, 0 to 40 min., linear gradient)

Example 1 Isolation of AB-1704 Strain

One loopful of the slant culture (0.5% of soluble starch, 0.5% ofglucose, 0.1% of fish meat extract (manufactured by Wako Pure ChemicalIndustries, Ltd.), 0.1% of yeast extract (manufactured by Oriental YeastCo., Ltd.), 0.2% of NZ-case (manufacured by Humko Sheffield ChemicalCo.), 0.2% of sodium chloride, 0.1% of calcium carbonate and 1.6% ofagar (manufactured by Wako Pure Chemical Industries, Ltd.)) of thestrain isolated from the soil was inoculated into a 65 mL test tubecontaining 7 mL of a seed medium (2.0% of soluble starch, 1.0% ofglucose, 0.5% of polypeptone (manufactured by Nihon Pharmaceutical Co.,Ltd.), 0.5% of yeast extract (manufactured by Oriental Yeast Co., Ltd.)and 0.1% of calcium carbonate), and it was cultured at 28° C. for threedays in a rotary shaker to give a seed culture broth.

Further, 0.5 mL of the seed culture broth was inoculated into a 65 mLtest tube containing 7 mL of a production medium (2.0% of solublestarch, 1.0% of glucose, 0.5% of polypeptone (manufactured by NihonPharmaceutical Co., Ltd.), 0.5% of yeast extract (manufactured byOriental Yeast Co., Ltd.), and 0.1% of calcium carbonate), and it wascultured at 28° C. for three days in a rotary shaker. Next, a 25 mg/mLsolution of the substrate 11107B substance in ethanol was prepared, and0.2 mL of the solution was added to the culture. After addition, it wasshaken at 28° C. for 48 hours to carry out conversion reaction. Afterthe reaction, the reaction mixture was analyzed by HPLC under thefollowing analytic HPLC condition (a), to give AB-1704 strain (FERMBP-8551) by which the 11107D substance was formed in thereaction-mixture.

Analytic HPLC Condition (a)

Column: CAPCELL PAK C18 SG120 φ4.6 mm×250 mm (manufactured by SHISEIDOCO.,)

Temperature: 40° C.

Flow rate: 1 mL/min.

Detection: 240 nm

Eluent: acetonitrile/0.15% potassium dihydrogenphosphate (pH 3.5) (3:7to 5:5, v/v, 0 to 18 minutes, linear gradient), acetonitrile/0.15%potassium dihydrogenphosphate (pH3.5) (5:5to85:15, v/v, 18to22minutes,linear gradient)

Retention time: 11107D substance 9.9 min., 11107B substance 19.4 min.

Example 2 Isolation of A-1544 Strain and A-1545 Strain

One loopful of the slant culture (yeast-malt agar) of the strainisolated from the soil was inoculated into a 250 mL Erlenmeyer flaskcontaining 20 mL of a seed medium (2.4% of soluble starch, 0.1% ofglucose, 0.5% of soybean meal (ESUSAN-MEAT manufactured by AjinomotoCo., Ltd.), 0.3% of beef extract (manufactured by Difco), 0.5% of yeastextract (manufactured by Difco), 0.5% of triptone-peptone (manufacturedby Difco), and 0.4% of calcium carbonate), and it was cultured at 28° C.for three days in a rotary shaker to give a seed culture broth.

Further, 0.6 mL of the seed culture broth was inoculated into a 500 mLErlenmeyer flask containing 60 mL of a production medium (2.0% ofsoluble starch, 2.0% of glucose, 2.0% of soybean meal (ESUSAN-MEATmanufactured by Ajinomoto Co., Ltd.), 0.5% of yeast extract(manufactured by Oriental Yeast Co., Ltd.), 0.25% of sodium chloride,0.32% of calcium carbonate, 0.0005% of copper sulfate, 0.0005% ofmanganese chloride, 0.0005% of zinc sulfate, pH 7.4 beforesterilization), and it was cultured at 28° C. for four days in a rotaryshaker. Each 2 mL of the resulting culture was dispensed into 15 mL testtubes. Next, a 20 mg/mL solution of the substrate 11107B substance indimethyl sulfoxide was prepared, and 0.05 mL of the solution was added.After the addition, it was shaken at 28° C. for 23 hours to carry outconversion. After the reaction, the reaction mixture was analyzed byHPLC under the following analytic HPLC condition (b) to give A-1544strain (FERMBP-8446) and A-1545 strain (FERM BP-8447) by which the11107D substance was formed in the reaction mixture.

Analytic HPLC Condition (b)

Column: CAPCELLPAKC18 SG120 φ4.6 mm×250 mm (manufactured by SHISEIDOCO.,)

Temperature: 40° C.

Flow rate: 1 mL/min.

Detection: 240 nm

Eluent: acetonitrile/water (50:50, v/v) Isocratic Retention time: 11107Bsubstance 7.2 min., 11107D substance 3.6 min.

Example 3 Conversion by AB-1704 Strain in a Flask Scale

One loopful of the slant culture (0.5% of soluble starch, 0.5% ofglucose, 0.1% of fish meat extract (manufactured by Wako Pure ChemicalIndustries, Ltd.), 0.1% of yeast extract (manufactured by Oriental YeastCo., Ltd.), 0.2% of NZ-case (manufacured by Humko Sheffield ChemicalCo.), 0.2% of sodium chloride, 0.1% of calcium carbonate, and 1.6% ofagar (manufactured by Wako Pure Chemical Industries, Ltd.)) ofStreptomyces sp. AB-1704 strain (FERM BP-8551) isolated from the soilwas inoculated into a 500 mL Erlenmeyer flask containing 100 mL of aseed medium (2.0% of soluble starch, 1.0% of glucose, 0.5% ofpolypeptone (manufactured byNihon Pharmaceutical Co., Ltd.), 0.5% ofyeast extract (manufactured by Oriental Yeast Co., Ltd.) and 0.1% ofcalcium carbonate), and it was cultured at 28° C. for three days on arotary shaker to give a seed culture broth. Further, 2 mL of the seedculture broth was inoculated into each of 150 Erlenmeyer flasks having acapacity of 500 mL and containing 100 mL of a production medium (2.0% ofsoluble starch, 1.0% of glucose, 0.5% of polypeptone (manufactured byNihon Pharmaceutical Co., Ltd.), 0.5% of yeast extract (manufactured byOriental Yeast Co., Ltd.) and 0.1% of calcium carbonate), and it wascultured at 28° C. for two days on a rotary shaker.

A 20 mg/mL solution of the substrate 11107B substance in ethanol wasprepared, and each 0.44 mL of the solution was added to the resultingculture (100 mL/500 mL Erlenmeyer flask, 150 flasks). After theaddition, it was shaken at 28° C. for 9 hours to conduct conversionreaction. After the completion of the reaction, the cultures werecollected and separated into the culture supernatant and the mycelia bycentrifugation at 2700 rpm for 10 minutes. The mycelia was extractedwith 5 L of methanol and filtered to give the methanol extract solution.This methanol extract solution was evaporated to remove methanol,combined with the culture supernatant and extracted with 10 L of ethylacetate. The resulting ethyl acetate solution was evaporated to give2090 mg of a crude active fraction. The crude active fraction wasdissolved in 4 mL of a mixture of tetrahydrofuran-methanol (1:1, v/v)and 6 mL of a 50% aqueous solution of acetonitrile, subjected to ODScolumn chromatography (manufactured by YMC Co., ODS-AM 120-S50 φ3.6cm×43 cm) and eluted with a 40% aqueous solution of acetonitrile. Aneluted fraction from 336 mL to 408 mL was concentrated to dryness underreduced pressure to give 560 mg of the residue. Further, the residue wasdissolved in 10 mL of a 50% aqueous methanol solution, subjected to ODScolumn chromatography (manufactured by YMC Co., ODS-AM 120-S50 φ3.6cm×40 cm) and eluted with a 50% aqueous solution of methanol. An elutedfraction from 1344 mL to 1824 mL was concentrated to dryness underreduced pressure to give 252 mg of 11107D substance. Example 4Conversion by A-1545 strain in a flask scale

One loopful of the slant culture (yeast-maltagar) of A-1545 strain (FERMBP-8447) was inoculated into a 250 mL Erlenmeyer flask containing 25 mLof a seed medium (2.0% of soluble starch, 2.0% of glucose, 2.0% ofsoybean meal (ESUSAN-MEAT manufactured by Ajinomoto Co., Ltd.), 0.5% ofyeast extract (manufactured by Difco), 0.25% of sodium chloride, and0.32% of calcium carbonate, pH 7.4 before sterilized), and it wascultured at 28° C. for two days in a rotary shaker to give a seedculture broth. Each 0.75 mL of the broth was dispensed into 2 mL serumtubes (manufactured by Sumitomo Bakelite Co., Ltd.), and an equal amountof a 40% aqueous solution of glycerol was added. After stirring, it wasfrozen at −70° C. to give a frozen seed. The frozen seed was melted,0.25 mL thereof was inoculated into a 250 mL Erlenmeyer flask containing25 mL of a seed medium (2.0% of soluble starch, 2.0% of glucose, 2.0% ofsoybean meal (ESUSAN-MEAT manufactured by Ajinomoto Co., Ltd.), 0.5% ofyeast extract (manufactured by Oriental Yeast Co., Ltd.), 0.25% ofsodium chloride and 0.32% of calcium carbonate, pH 7.4 beforesterilized), and it was cultured at 28° C. for two days on a rotaryshaker to give a seed culture broth. Further, the seed culture broth(0.5 mL) was inoculated into a 500 mL Erlenmeyer flask containing 100 mLof a production medium (2.0% of soluble starch, 2.0% of glucose, 2.0% ofsoybean meal (ESUSAN-MEAT manufactured by Ajinomoto Co., Ltd.), 0.5% ofyeast extract (manufactured by Oriental Yeast Co., Ltd.), 0.25% ofsodium chloride, and 0.32% of calcium carbonate, pH 7.4 beforesterilized), and it was cultured at 28° C. for three days on a rotaryshaker.

Each of the resulting culture broths (100 mL/500 mL Erlenmeyer flask, 10flasks) was subjected to centrifugation at 3000 rpm for 10 minutes tocollect microorganism cells, and the cells were suspended into 100 mL ofa 50 mM phosphate buffer solution (pH 6.0). Next, a 100 mg/mL solutionof the substrate 11107B substance in dimethyl sulfoxide was prepared,and each 1 mL of the solution was added. After the addition, it wasshaken at 28° C. for 24 hours to conduct conversion reaction. After thecompletion of the reaction, the reaction solutions were collected andseparated into the supernatant and the mycelia by centrifugation at 5000rpm for 20 minutes. The supernatant was extracted with 1 L of ethylacetate. The mycelia was extracted with 500 mL of methanol and thenfiltered to obtain a methanol extract. The methanol extract wasevaporated to remove methanol and extracted with 1 L of ethyl acetate.Each of the ethyl acetate layers was washed with water, dried anddehydrated over anhydrous sodium sulfate, and the combined layers wereevaporated to give 937 mg of a crude fraction. The crude fraction wassubjected to silica gel column chromatography (Kiesel gel 60, 50 g) andeluted with 1200 mL of a mixture of ethyl acetate and n-hexane (90:10;v/v) to obtain 234 mg of a fraction containing the 11107D substance. Theresulting active fraction was subjected to preparative high performanceliquid chromatography (HPLC) under the following preparative HPLCcondition (C), and the resulting eluate was analyzed by HPLC under thefollowing analytic HPLC condition (c). The solvent was removed from thefraction containing the 11107D substance thus obtained, to give 80 mg ofthe 11107D substance.

Preparative HPLC Condition (C)

Column: CAPCELL PAK C18 UG120 φ30 mm×250mm (manufactured by SHISEIDOCo.)

Flow rate: 20 mL/min.

Detection: 240 nm

Eluent: acetonitrile/water (30:70, v/v) isocratic

Analytic HPLC Condition (c)

Column: CAPCELLPAKC18 SG120 φ4.6 mm×250 mm (manufactured by SHISEIDOCo.)

Temperature: 40° C.

Flow rate: 1 mL/min.

Detection: 240 nm

Eluent: acetonitrile/water (35:65, v/v) isocratic

Retention time: 11107D substance 7.8 min.

Example 5 Conversion by A-1544 Strain in a Flask Scale

Each of cultures of A-1544 strain (FERMBP-8446) (100 mL/500 mLErlenmeyer flask, 10 flasks) obtained by a similar method as describedin Example 4 was subjected to centrifugation at 3000 rpm for 10 minutesto collect microorganism cells, and the cells were suspended into 100 mLof a 50 mM phosphate buffer solution (pH 6.0). Next, a 100 mg/mLsolution of the substrate 11107B in dimethyl sulfoxide was prepared, andeach 1 mL of the solution was added. After the addition, it was shakenat 28° C. for 24 hours to run a conversion reaction. After thecompletion of the reaction, the reaction solutions were collected andseparated into the supernatant and the mycelia by centrifugation at 5000rpm for 20 minutes. The supernatant was extracted with 1 L of ethylacetate. The mycelia was extracted with 500 mL of acetone, and thenfiltered to give an acetone extract. The acetone extract solution wasevaporated to remove acetone, and then the residue was extracted with 1L of ethyl acetate. Each of the ethyl acetate layers was washed withwater, dried and dehydrated over anhydrous sodium sulfate, and thecombined layers were evaporated to give 945 mg of a crude fraction. Thecrude fraction was subjected to silica gel column chromatography (Kieselgel 60, 50 g), eluted with 100 mL of a mixture of ethyl acetate andn-hexane (50:50; v/v), 200 mL of a mixture of ethyl acetate and n-hexane(75:25; v/v) and a mixture (600 mL) of ethyl acetate and n-hexane(90:10; v/v), to give 463mg of a fraction containing the 11107Dsubstance. The resulting fraction was subjected to preparative highperformance liquid chromatography (HPLC) under the preparative HPLCcondition (C) described in Example 4 and the resulting eluate wasanalyzed by HPLC under the analytic HPLC condition (c) described inExample 4. The solvent was removed from the fraction containing 11107Dsubstance thus obtained, to give 304 mg of the 11107D substance.

Example 6 Isolation of F-1529 Strain and F-1530 Strain

One loopful of the slant culture (potato dextrose agar) of the strainisolated from the soil was inoculated into a 250 mL Erlenmeyer flaskcontaining 20 mL of a seed medium (2.0% of potato starch, 1.0% ofglucose, 2.0% of a soybean meal (ESUSAN-MEAT manufactured by AjinomotoCo., Ltd.), 0.1% of potassium dihydrogenphosphate and 0.05% of magnesiumsulfate heptahydrate), and it was cultured at 25° C. for three days on arotary shaker to give a seed culture broth. Further, 0.6 mL of the seedculture broth was inoculated into 500 mL Erlenmeyer flask containing 60mL of a production medium (2.0% of potato starch, 1.0% of glucose, 2.0%of a soybean meal (ESUSAN-MEAT manufactured by Ajinomoto Co., Ltd.),0.1% of potassium dihydrogenphosphate and 0.05% of magnesium sulfateheptahydrate), and it was cultured at 28° C. for four days on a rotaryshaker. 2 mL of the resulting culture broth was dispensed in each of 15mL test tubes. Each test tube was subjected to centrifugation at 3000rpm for 5 minutes to collect microorganism cells and then suspended in 2mL of a 50 mM phosphate buffer solution (pH 7.0). Then, a 20 mg/mLsolution of the substrate 11107B substance in dimethyl sulfoxide wasprepared, and 0.05 mL of the solution was added. After the addition, itwas shaken at 28° C. for 23 hours to conduct a hydroxylation reaction.After the reaction, the reaction mixture was analyzed by HPLC under theanalytic condition (c) described in Example 4 to give F-1529 strain(FERM BP-8547) and F-1530 strain (FERM BP-8548) which both had the peakof the 11107D substance in HPLC.

Example 7 Conversion by F-1529 Strain in a Flask Scale

One loopful of the slant culture (potato dextrose agar) of Mortierellasp. F-1529 strain (FERM BP-8547) was inoculated into a 250 mL Erlenmeyerflask containing 25 mL of a seed medium (2.0% of potato starch, 1.0% ofglucose, 2.0% of a soybean meal (ESUSAN-MEAT manufactured by AjinomotoCo., Ltd.), 0.1% of potassium dihydrogenphosphate and 0.05% of magnesiumsulfate heptahydrate), and it was cultured at 25° C. for two days on arotary shaker to give a seed culture broth. Further, 0.6 mL of the seedculture broth was inoculated into each of a 500 mL Erlenmeyer flaskcontaining 60 mL of a production medium (2.0% of potato starch, 1.0% ofglucose, 2.0% of a soybean meal (ESUSAN-MEAT manufactured by AjinomotoCo., Ltd.), 0.1% of potassium dihydrogenphosphate and 0.05% of magnesiumsulfate heptahydrate), and it was cultured at 25° C. for three days on arotary shaker.

Each resulting culture broth (60 mL/500 mL Erlenmeyer flask, 18 flasks)was subjected to centrifugation conducted out at 3000 rpm for 5 minutesto collect microorganism cells and then suspended in 60 mL of a 50 mMphosphate buffer solution (pH 7.0). Next, a 100 mg/mL solution of thesubstrate 11107B substance in dimethyl sulfoxide was prepared, and each0.6 mL of the solution was added. After the addition, it was shaken at25° C. for 22 hours to conduct conversion reaction. After the completionof the reaction, the culture broth was separated into the supernatantand the mycelia by centrifugation at 5000 rpm for 20 minutes. Thesupernatant was extracted with 1 L of ethyl acetate. The mycelia wasextracted with 500 mL of acetone and filtered to give an acetone extractsolution. The acetone extract solution was evaporated to remove acetoneand then extracted with 1 L of ethyl acetate. The ethyl acetate layerswere respectively washed with water, dehydrated and dried over anhydroussodium sulfate and then combined with each other and evaporated, to give1.21 g of a crude fraction including the 11107D substance. The crudefraction including the 11107D substance was subjected to silica gelcolumn chromatography (Kiesel gel 60, 50 g), eluted with 1200 mL of amixture of ethyl acetate and n-hexane (90:10; v/v), to give 369 mg of afraction containing the 11107D substance.

The resulting fraction was subjected to preparative high performanceliquid chromatography (HPLC) under the preparative HPLC condition (C)described in Example 4 to give an eluted fraction containing the 11107Dsubstance. Then, the solvent was removed to give 180 mg of the 11107Dsubstance.

Example 8 Conversion by F-1530 Strain in a Flask Scale

One loopful of the slant culture (potato dextrose agar) of Mortierellasp. F-1530 strain (FERM BP-8548) was inoculated into a 250 mL Erlenmeyerflask containing 25 mL of a seed medium (2.0% of potato starch, 1.0% ofglucose, 2.0% of a soybean meal (ESUSAN-MEAT manufactured by AjinomotoCo., Ltd.), 0.1% of potassium dihydrogenphosphate and 0.05% of magnesiumsulfate heptahydrate), and it was cultured at 25° C. for two days on arotary shaker to give a seed culture broth. Further, 0.6 mL of the seedculture broth was inoculated into a 500 mL Erlenmeyer flask containing60 mL of a production medium (2.0% of potato starch, 1.0% of glucose,2.0% of a soybean meal (ESUSAN-MEAT manufactured by Ajinomoto Co.,Ltd.), 0.1% of potassium dihydrogenphosphate and 0.05% of magnesiumsulfate heptahydrate), and it was cultured at 25° C. for three days on arotary shaker.

Each obtained culture broth (60 mL/500 mL Erlenmeyer flask, 18 flasks)was subjected to centrifugation conducted at 3000 rpm for 5 minutes tocollect microorganism cells and then suspended in 60 mL of a 50 mMphosphate buffer solution (pH 7.0). Next, a 100 mg/mL solution of thesubstrate 11107B substance in dimethyl sulfoxide was prepared, and each0.6 mL of the solution was added. After the addition, it was shaken at25° C. for 22 hours to conduct conversion reaction. After the completionof the reaction, the culture broth was separated into the supernatantand the mycelia by centrifugation at 5000 rpm for 20 minutes. Thesupernatant was extracted with 1 L of ethyl acetate. The mycelia wasextracted with 500 mL of acetone and filtered to give an acetone extractsolution. The acetone extract solution was evaporated to remove acetoneand then extracted with 1 L of ethyl acetate. The ethyl acetate layerswere respectively washed with water, dehydrated and dried usinganhydrous sodium sulfate and then combined with each other andevaporated to give 0.89 g of a crude fraction including the 11107Dsubstance. The crude fraction including the 11107D substance wassubjected to silica gel column chromatography (Kiesel gel 60, 50 g),eluted with 1200 mL of a mixture of ethyl acetate and n-hexane (90:10;v/v) and then with 500 mL of ethyl acetate, to give 163mg of a crudefraction containing the 11107D substance. The resulting fraction wassubjected to preparative high performance liquid chromatography (HPLC)under the HPLC condition (C) described in Example4 to give an elutedfraction containing the 11107D substance. Then, the solvent was removedto give 30 mg of the 11107D substance.

Example 9 Isolation of AB-1896 Strain

One loopful of the slant culture (0.5% of soluble starch, 0.5% ofglucose, 0.1% of fish meat extract (manufactured by Wako Pure ChemicalIndustries, Ltd.), 0.1% of yeast extract (manufactured by Oriental YeastCo., Ltd.), 0.2% of NZ-case (manufactured by Humko Sheffield ChemicalCo.), 0.2% of sodium chloride, 0.1% of calcium carbonate, and 1.6% ofagar (manufactured by Wako Pure Chemical Industries, Ltd.)) of thestrain isolated from the soil was inoculated into a 65 mL test tubecontaining 5 mL of a seed medium (2.0% of soluble starch, 1.0% ofglucose, 0.5% of polypeptone (manufactured by Nihon Pharmaceutical Co.,Ltd.), 0.5% of yeast extract (manufactured by Oriental Yeast Co., Ltd.),and 0.1% of calcium carbonate), and it was cultured at 28° C. for tendays in a rotary shaker to give a seed culture broth. Further, 0.1 mL ofthe seed culture broth was inoculated into a 65 mL test tube containing5 mL of a production medium (2.0% of soluble starch, 1.0% of glucose,0.5% of polypeptone (manufactured by Nihon Pharmaceutical Co., Ltd.),0.5% of yeast extract (manufactured by Oriental Yeast Co., Ltd.), and0.1% of calcium carbonate), and it was cultured at 28° C. for three daysin a rotary shaker. Next, a 40 mg/mL solution of the substrate 11107Bsubstance in ethanol was prepared, and 0.05 mL of the solution was addedto the culture. After addition, it was shaken at 28° C. for 24 hours tocarry out hydroxylation reaction. After the reaction, the culture brothwas subjected to HPLC analysis according to the following analyticcondition (d) to give AB-1896 strain by which the 11107D substance wasformed.

Analytic HPLC Condition (d)

Column: UNISON UK-C18, φ4.6 mm×50 mm (manufactured by Imtakt)

Temperature: 30° C.

Flow rate: 2 mL/min.

Detection: 240 nm

Eluent: water/acetonitrile/formic acid (1000:10:1 to 10:1000:1, v/v/v, 0to 4 minutes, linear gradient)

Retention time: 11107D substance 2.5 min.

Example 10 Conversion by AB-1896 Strain in a Test-Tube Scale

One loopful of the slant culture (0.5% of soluble starch, 0.5% ofglucose, 0.1% of fish meat extract (manufactured by Wako Pure ChemicalIndustries, Ltd.), 0.1% of yeast extract (manufactured by Oriental YeastCo., Ltd.), 0.2% of NZ-case (manufacured by Humko Sheffield ChemicalCo.), 0.2% of sodium chloride, 0.1% of calcium carbonate, and 1.6% ofagar (manufactured by Wako Pure Chemical Industries, Ltd.)) of AB-1896strain was inoculated into a 65 mL test tube containing 5 mL of a seedmedium (2.0% of soluble starch, 1.0% of glucose, 0.5% of polypeptone(manufactured by Nihon Pharmaceutical Co., Ltd.), 0.5% of yeast extract(manufactured by Oriental Yeast Co., Ltd.) and 0.1% of calciumcarbonate), and it was cultured at 28° C. for 10 days on a rotary shakerto give a seed culture broth. Further, 0.1 mL of the seed culture brothwas inoculated into a 65 mL test tube containing 5 mL of a productionmedium (2.0% of soluble starch, 1.0% of glucose, 0.5% of polypeptone(manufactured by Nihon Pharmaceutical Co., Ltd.), 0.5% of yeast extract(manufactured by Oriental Yeast Co., Ltd.), and 0.1% of calciumcarbonate), and it was cultured at 28° C. for three days in a rotaryshaker.

A 40 mg/mL solution of the substrate 11107B substance in ethanol wasprepared, and 0.05 mL of the solution was added to the resulting culturebroth (5 mL/65 mL test tube). After addition, it was shaken at 28° C.for 24 hours to carry out hydroxylation reaction. 3 mL of the resultingculture broth was separately collected, 2 mL of 1-butanol was addedthereto, shaken, and then centrifuged at 3000 rpm for 10 minutes. Theresulting supernatant was removed, to prepare a 2mL of methanol solutionof the residue. It was subjected to HPLC analysis according to thefollowing analytic conditions (e) and (f) to confirm that the 11107Dsubstance was formed in the reaction mixture.

Analytic HPLC Condition (e)

Column: UNISON UK-Cl8, φ4.6 mm×50 mm (manufactured by Imtakt)

Temperature: 40° C.

Flow rate: 2 mL/min.

Detection: 240 nm

Eluent: acetonitrile/0.01% trifluoroacetic acid (2:8 to 5:5, v/v, 0 to10 minutes, linear gradient)

Retention time: 11107D substance 6.1 min.

Analytic HPLC Condition (f)

Column: UNISON UK-C18, φ4.6 mm×50 mm (manufactured by Imtakt)

Temperature: 40° C.

Flow rate: 2 mL/min.

Detection: 240 nm

Eluent: methanol/0.01% trifluoroacetic acid (4:6 to 7:3, v/v, 0 to 10minutes, linear gradient)

Retention time: 11107D substance 6.1 min.

1. A method of producing the macrolide compound 11107D represented by the formula (II):

wherein the macrolide compound 11107D is produced from the macrolide compound 11107B represented by the formula (I):

by a biological transformation method, which comprises the following processes (A) and (B): (A) a process of incubating the macrolide compound 11107B represented by the formula (I) in the presence of a strain having an ability of conducting the above-mentioned biological transformation method and belonging to the genus Mortierella, the genus Streptomyces or the family Micromonosporaceae or a preparation of its cultured mycelia; and (B) a process of collecting the macrolide compound 11107D represented by the formula (II) from the incubated solution obtained in the step (A).
 2. The production method according to claim 1, wherein the strain belonging to the genus Mortierella is Mortierella sp. F-1529strain (FERMBP-8547) or F-1530 strain (FERMBP-8548).
 3. The production method according to claim 1, wherein the strain belonging to the genus Streptomyces is Streptomyces sp. AB-1704 strain (FERM BP-8551), A-1544 strain (FERM BP-8446) or A-1545 strain (FERM BP-8447).
 4. The production method according to claim 1, wherein the strain belonging to the family Micromonosporaceae is AB-1896 strain (FERM BP-8550).
 5. Streptomyces sp. AB-1704 strain (FERM BP-8551) having the ability of transforming the macrolide compound 11107B represented by the above formula (I) into the macrolide compound 11107D represented by the above formula (II).
 6. Mortierella sp. F-1529strain (FERMBP-8547) or F-1530 strain (FERM BP-8548) having the ability of transforming the macrolide compound 11107B represented by the above formula (I) into the macrolide compound 11107D represented by the above formula (II).
 7. AB-1896 strain (FERM BP-8550) having the ability of transforming the macrolide compound 11107B represented by the above formula (I) in to the macrolide compound 11107D represented by the above formula (II). 